An epidermal serine sensing system for skin healthcare
Materials
All chemical reagents were purchased from InnoChem (China) unless otherwise stated.
Preparation of the serine sensor
Initially, the carbon ink (Jujo Printing Supplies & Technology, Japan) was spin-coated onto a clean polyethylene terephthalate (PET) with a thickness of 0.08 mm (Huanan Xiangcheng Technology, China) as the substrate layer. The spin-coating process was conducted twice at 3000 rpm for 15 s with an acceleration rate of 200 rpm/s. After drying, the prepared carbon electrode was cut into the pre-designed shape using a laser cutting machine (Suzhou Inngu Laser, China). The carbon electrode was then activated by cyclic voltammetry (CV) scanning in 0.5 M H2SO4 (Fengchuan, China) for 60 segments, ranging from −1.2 to 1 V at a scan rate of 500 mV/s. The activated carbon electrode was further modified with electrochemically synthesized PBNPs as the active layer by CV scanning in a mixed solution containing 3 mM FeCl3, 3 mM K3Fe(CN)6, 0.1 M HCl (Fengchuan, China), and 0.1 M KCl for 20 cycles (from −0.2 to 0.6 V at a scan rate of 50 mV/s). This process was repeated three times to ensure stable and optimal redox signals. As such, the sensor would exhibit a stable 115 μA LSV current peak in 0.1 M KCl. After that, the electrode was rinsed with distilled water and immersed in a solution comprising 0.1 M HCl and 0.1 M KCl for repeated CV scans (from −0.2 to 0.6 V at a scan rate of 50 mV/s) until a stable response was achieved for the PBNPs layer.
The preparation process of the MIP layer was carried out in a 0.1 M PBS solution containing 5 mM serine, 12.5 mM APBA, and 37.5 mM pyrrole. The MIP layer was electrochemically synthesized on the electrode via CV deposition (0−1 V, 5 cycles, 50 mV/s). The electrode was then soaked in an acetic acid/methanol mixture (7:3 v/v) for 12 h to extract the serine molecules. Following this, the prepared MIP-based electrode was immersed in 0.1 M KCl and conducted CV scans (from −0.2 V to 0.6 V at a scan rate of 50 mV/s) until a stable response was achieved. For the preparation of the NIP-based electrode, the same procedure was followed, with the exception that no template was added to the polymerization solution.
To fabricate the Ag/AgCl reference electrode, a silver layer was deposited on the substrate electrode surface using a multi-current steps technology in an electrolyte solution composed of 0.25 M silver nitrate, 0.75 M sodium thiosulfate, and 0.5 M sodium bisulfite. The electrochemical deposition parameters are as follows: −0.01 mA for 150 s, −0.02 mA for 50 s, −0.05 mA for 50 s, −0.08 mA for 50 s, and −0.1 mA for 350 s. Subsequently, 0.1 M FeCl3 solution was dripped onto the Ag surface for 20 s, and then immediately rinsed distilled water. A cocktail for reference potential stabilization was then drop-coated onto the Ag/AgCl electrode surface (3 μL), which was a mixture of 250 mg NaCl, 395.5 mg 79.1 mg polyvinyl butyral (PVB), 10 mg Poly (ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (F127, purchased from Sigma-Aldrich, USA), and 1 mg MWCNTs (XFNANO Materials, China) dispersed uniformly in 5 mL methanol.
Preparation of the porous PVA hydrogel
Initially, PVA (Mw ≈ 89,000, purchased from Sigma-Aldrich, USA) was dissolved in water at a weight ratio of 1:10 and heated at 90 °C for 2 h to obtain a homogeneous and transparent PVA solution. Separately, KOH was dissolved in water at a weight ratio of 1:5. Under continuous stirring, 14 g KOH solution was gradually added dropwise to 10 g PVA solution, and then 2.6 g of sucrose was dissolved into this mixture to form the precursor solution for the hydrogel. Next, 15 g precursor solution was poured into a petri dish (9 cm in diameter) and placed in a vacuum desiccator to remove excess water and facilitate cross-linking. The hydrogel was then immersed in deionized water to eliminate the sucrose template and excess KOH until it reached a neutral pH. Following this, the porous PVA hydrogel was cut into a desired shape and stored in 0.1 M KCl solution for subsequent use.
Characterization of the serine sensor
All in vitro characterizations of prepared sensors were performed through an electrochemical workstation (CHI760E) in the solution of 0.1 M KCl or the PVA hydrogel containing 30 μL. For the MIP-based serine sensor, LSV analysis was performed across varying concentrations of serine. The LSV parameters were set as follows: sweep voltage from −0.2 to 0.4 V, scan rate of 50 mV/s, quiet time of 2 s, and sensitivity of 1 × 10−4 A/V. Prior to each LSV scan, an incubation period of 5 minutes was conducted to ensure that the sensor reached the quasi-steady state. The selectivity testing for the serine sensor was performed in a series of common interferent species solutions (3 mM serine, 0.06 mM Phe, 0.16 mM Tyr, 0.16 mM Glu, 1.2 mM Gly, 0.8 mM His, 1.8 mM urea, 4.5 mM lactic acid, 0.5 mM uric acid).
The surface morphology of the aforementioned electrodes in different preparation steps was characterized by Field Emission Scanning Electron Microscopy (FSEM, Hitachi SU8020, Japan), including the carbon electrode, the MIP-based electrode before and after template removal, as well as the NIP electrode before and after template removal.
Fabrication and assembly of the serine sensing patch
The laser-cutting device was employed to process the sensing chamber and two encapsulation layers of the sensor. All laser-engraved patterns were pre-designed using AutoCAD 2020. Specifically, a waterproof tape was cut into rounded rectangles of appropriate size as the encapsulation layer for the upper and lower layers to effectively inhibit water loss in the hydrogel. a chamber for placing the hydrogel was penetrated with a through-hole (diameter = 0.95 mm, thickness = 0.32 mm) in a double-side tape. All layers were vertically assembled, from top to bottom, which was the top encapsulation layer, the sensing chamber, the serine sensing electrode, and the bottom encapsulation layer. The top encapsulation layer can be torn off and attach the patch to the skin when used.
Circuit design of the handheld serine tester
The circuit module of the serine sensing tester comprises four primary parts: First, the microcontroller unit (MCU) serves as the core control unit of the tester system. Second, the power management system ensures a stable power supply. Finally, the three-electrode circuit and the screen control circuit are respectively responsible for specific signal processing and display control functions. Through precise connection and configuration, all components collaborate seamlessly to achieve LSV potential waveform signal readout, processing, transmission, and display.
As the core of the MCU control circuit, an STM32F301K8U6 chip is capable of delivering high-performance digital signal processing and meeting the demands of multiple peripheral interfaces. For LSV scanning, a predefined excitation potential waveform was applied across the working electrode and the reference electrode through a 12-bit digital-to-analog converter (DAC) built in MCU. The resulting current signals were then converted into voltage signals by a transimpedance amplifier within the circuit. Then these converted voltage signals were captured by an integrated 12-bit Analog-to-Digital Converter (ADC) built in the MCU, constructing a relationship curve between the excitation voltage and current signals. The MCU performed baseline correction, filtering, and smoothing on the raw LSV data. By utilizing the measured LSV curve in the blank solution (0.1 M KCl) as a calibration standard, subsequent LSV curves of serine levels were calibrated. Based on the calibrated data and a preset algorithm, the MCU converted the processed data into serine concentration values.
The MCU transmitted the epidermal serine level result to a liquid crystal display (LCD) screen through a high-speed serial peripheral interface (SPI) protocol. The screen is a 1.83-inch IPS color LCD screen with a power supply voltage of 3.3 V and a resolution of 240 x 280. It is driven by a NV3030B chip to ensure clear display content and bright colors.
The power management system is divided into a regulated power supply circuit and a charging management circuit. The regulated power supply module uses a rechargeable 3.7 V lithium-ion polymer battery with appropriate capacity and size as the main power supply. The battery voltage was converted into stable 3.3 V digital and analog power supplies through a low dropout linear regulator (662 K) to reduce potential interference from digital circuits on analog signals. The core of the charging management circuit is a power management chip (4056 A), which uses the power transistors inside the chip to perform constant current and constant voltage charging on the battery.
Program design of the handheld serine tester
The software component was designed in accordance with the functional modules of the hardware circuitry, utilizing the Keil µVision 5 integrated development environment and the C programming language for software editing, compilation, and real-time debugging. The system’s relevant programs encompass a MAIN program, an A/D conversion subroutine, a D/A conversion subroutine, a data processing subroutine, and an LCD display driver subroutine. The programs developed on the personal computer were written into the MCU by the ST-Link. The MCU was connected to the ST-Link through pins including power, ground, clock, and data lines (VCC, GND, SWCLK, SWDIO). The ST-Link was configured as the debugger in the Keil µVision 5, and the designed program was burned into the Flash memory of the MCU.
Shell design of the handheld serine tester
The shell of the tester was designed by Solidworks and made by machining, which includes an upper cover plate and a bottom back plate. Screw fixing points were reserved around the plates for fixing each other. A charging port, a button port, a sliding switch interface, and a patch socket were reserved on the shell. In addition, the hollow area in the middle of the upper cover plate could place the LCD display screen. The whole tester’s shell was elaborately designed to ensure a suitable layout of internal components while maintains the overall compactness and portability, which is convenient for users to carry and use. This customized shell is fully parameterized, allowing for easy scaling and adjustment of tolerances according to user needs to achieve optimal fit.
Ethics
The human subject experiments strictly adhere to the ethical guidelines set forth by institutional or national research committees, as well as the Declaration of Helsinki of the World Medical Association. This particular study has received ethical approval from the Human Study Ethics Committee of Beijing Forestry University (Approval No. BJFUPSY-2024-049). All participants aged between 22 and 27 were recruited through verbal recruitments. Each participant was paid between 50 to 200 RMB (depending on the participation time) as the compensation for the test. Prior to testing, all participants provided written informed consent. The recruited subjects comprised seven healthy individuals and one patient with atopic dermatitis (AD). Furthermore, the seven healthy subjects recruited reported no history of allergies and presented with no skin lesion on the inner side of the forearm. The patient with AD, whose affected area on the neck does not exhibit severe erosion, exudation, or secondary infection.
Serine sensing system validation on human subjects
To validate the feasibility of our serine sensing system for detecting skin serine levels and analyzing skin conditions, we selected the inner side of subject’s forearm as the sampling site, the baseline characteristics of participants are detailed in Supplementary Table 3. The entire test was conducted in a relatively closed laboratory environment. During the experiment, the temperature was precisely controlled between 25 ± 1 °C, and the relative humidity was stably maintained within the range of 50 ± 5%. Considering the experiment primarily focuses on the short-term effect of a skincare product, we did not impose extremely strict restrictions on the specific time points for sampling within a day. Before participating in the test, subjects were strictly prohibited from using moisturizing, exfoliating, acid-containing, or other products that may affect the metabolism of the skin’s stratum corneum and the serine level in the test area within 12 h. In order to obtain the original surface information and serine level of the stratum corneum, no pre-treatment of the skin was required to prevent any mechanical of chemical damage to the stratum corneum and destruction of the skin surface metabolite components.
Before measurement, subjects were asked to sit quietly and rest for 30 minutes under the set environmental conditions to allow the skin to fully adapt to the environment and stabilize its baseline state. Then, subjects used the portable serine sensing system to measure serine levels in the various skin states in this work according to the operating instructions provided by us. A disposable patch was provided for each measurement.
When studying the robustness of the serine sensing system, we selected the forehead, the back of the hand, and the inner side of the forearm as the sampling sites. For tracking the treatment progress of the AD patient (three weeks), we chose the lesion area on the patient’s neck and the normal skin area surrounding the lesion as the test sites, the baseline characteristics of the patient are detailed in Supplementary Table 4. For the lesion area, obvious erythema and flakes were observed. The normal skin area surrounding the lesion was selected within a range of 2–3 cm from the edge of the lesion. A series of measurements were conducted during topical medication (at the 1st week and 3rd week) on both the lesion skin and adjacent normal skin areas. The specific timepoint for test was in the afternoon, and the method is the same as mentioned above, including environmental temperature and humidity control, skin treatment, and operational procedures. During this process, the improvement in the appearance of the lesion skin was recorded and photographed. All epidermal serine measurements using the serine sensing system were completed by the subjects themselves according to the operating instructions.
Colorimetry for system verification
All hydrogels within the patches after the previous measurements were collected and dried in 1.5 mL centrifuge tubes, and then stored at −25 °C. These hydrogels were further used to measure the average serine levels and the average AAs levels in their matrixes using the corresponding colorimetric assay kits. In order to extract serine/AAs from the hydrogels, dry hydrogel samples were immersed in 200 µL deionized water for 12 h to ensure that serine/AAs in hydrogel can be fully dissolved in water. The above extracts were directly used for subsequent colorimetric analysis. the L-Serine ELISA Kit (Immusmol, France) and the AAs assay kit (Solarbio, China) were used by following the steps on their instructions for the determinations of serine and AAs levels in the hydrogels, respectively.
The sampling of epidermal serine in the stratum corneum was conducted using a 3 M tape (Scotch book tape) cut to a 9 mm diameter. Initially, the tape was carefully placed on the skin surface without applying any additional pressure. A 100 g weight was then placed on the tape and maintained for 1 minute to ensure thorough adhesion and sampling. After the tape was smoothly peeled from the skin, the peeled tape was placed into a pre-labeled 1.5 mL centrifuge tube immediately and then stored at −25 °C. The collected tapes were further used to measure the serine levels using the colorimetric assay kits. Before colorimetric measurement, the tape was immersed in 200 μL of deionized water and subjected to 30 min of ultrasonic treatment to effectively extract the water-soluble serine molecules. The extracts were directly used for subsequent colorimetric analysis using the L-Serine ELISA Kit for the determination of serine and AAs levels in the tapes.
Statistical analysis
Statistical analyses were conducted using SPSS version 25 (IBM, USA) and Origin version 2024 (OriginLab, USA). We employed the least squares method of linear regression to establish the linear relationship between variables, which allowed us to calculate the Pearson correlation coefficient (r). Additionally, we used a two-tailed paired t-test to determine the statistical difference in epidermal serine levels before and after the application of essence. The result showed a statistically significant difference with a P-value of 0.003 (*P < 0.05). Prior to conducting the t-test, we assessed the normality of the data using the Shapiro-Wilk test to ensure the validity of our t-test. The result indicated that the data met the assumption of normality with a P-value of 0.338 (P > 0.05).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
link
